Temporal trends of the in vitro activity of tigecycline and comparator antibiotics against clinical aerobic bacterial isolates collected in Germany, 2006–2014: results of the Tigecycline Evaluation and Surveillance Trial (TEST)

Given the rapidly changing landscape of antimicrobial resistance, continuous monitoring of antimicrobial susceptibility in clinically relevant bacterial isolates plays an important role in the management of infectious diseases. The Tigecycline Evaluation and Surveillance Trial (TEST) is an ongoing worldwide surveillance programme monitoring the in vitro activity of tigecycline and a panel of representative comparator antibiotics. We report longitudinal susceptibility data on a large set of isolates (n=36,044) from clinically significant bacterial species collected in 25 microbiological laboratories from 2006 to 2014. Trends include a strong increase of carbapenem and levofloxacin resistance in Acinetobacter spp., and smaller increasing rates of ESBL-producing Escherichia coli and vancomycin-resistant enterococci. Across the reporting period, the tigecycline minimum inhibitory concentrations (MICs) at which 50% and 90% of isolates were inhibited remained stable and susceptibility rates were consistently high (93–100%) for all bacterial species.


Introduction
As the World Health Organization recently pointed out in its global report, antimicrobial resistance threatens the effective treatment of an increasing range of infectious diseases [1]. Patients with infections caused by multidrugresistant (MDR) bacteria are generally at elevated risk of unfavorable clinical outcomes and death [2]. In many settings and patient groups, standard antimicrobials are no longer considered adequate choices for empirical therapy of serious infections [3], [4]. A particularly concerning development is the rapid spread of MDR Gram-negative pathogens as infecting and colonizing organisms, which is mostly due to the expansion of genetic determinants associated with extended-spectrum beta-lactamases (ESBL) or carbapenemase production [5]. Tigecycline (Tygacil ® ; Pfizer Inc.) is a glycylcycline antibiotic [6] licensed for complicated intraabdominal infections (cIAI) and complicated skin/soft-tissue infections (cSSTI) since 2006. It exhibits activity against a broad spectrum of aerobic and anaerobic Gram-negative and Gram-positive bacteria including most MDR organsims of the critically important group of "ESCAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae) [7] such as Gram-negative bacteria producing ESBLs and/or carbapenemases, methicillinresistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE) [8]. P. aeruginosa is intrinsically resistant to tigecyline. Susceptibility to tigecycline may be reduced in bacteria of the Proteeae tribe, mostly mediated by overexpression of efflux pumps [9]. Acquired resistance has been described in isolates of A. baumannii, several Enterobacteriaceae species and Gram-positive cocci [8], [10]. In view of the fact that the number of MDR bacterial pathogens has increased over the past 20-30 years, tigecycline plays an important role in the management of complicated bacterial infections. Longitudinal monitoring of the antimicrobial activity of tigecycline is important in assessing the continued usefulness of this agent. As pathogen distribution and resistance patterns show substantial heterogeneity across countries, as evident for Europe from the data compiled by the EARS-Net [11], country-specific data on the tigecycline suceptibility is necessary and has been published for several geographic areas, including Germany [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. The Tigecycline Evaluation and Surveillance Trial (TEST) monitors the in vitro activity of tigecycline and a panel of representative comparator antibiotics against clinically relevant pathogens from microbiological laboratories worldwide [22]. This report provides susceptibility data of tigecycline and comparator agents collected as part of TEST [23] in Germany between the market introduction of tigecycline in 2006 until 2014, thus expanding earlier German data from TEST reported by Seifert and Dowzicky for the years 2004-2007 [24] to a more recent observation period. The present report focussed on (i) bacterial species which are frequently associated with resistance phenotypes and infections in severely ill patients and (ii) antimicrobials frequently used in severe infection and/or representing important groups of antibiotics. A full regularly updated data set of this study is available online via the TEST surveillance website [23].

Bacterial strains
This report includes data from clinical isolates collected by 25 German microbiological laboratories in the time period from 2006 to 2014. Some laboratories, however, did not participate for the entire study period covered in this report. At the beginning of TEST, each participating laboratory had to provide a minimum of 200 isolates per year. These were 135 isolates of Gram-negative pathogens comprising Acinetobacter spp. (n=10) and 65 isolates of Gram-positive pathogens comprising Enterococcus spp. (E. faecium and E. faecalis only; n=15), S. aureus (n=25), Streptococcus agalactiae (n=10) and Streptococcus pneumoniae (n=15). As of 2012, clinical isolates of two additional important genera, Citrobacter spp. [25] and Stenotrophomonas spp., were included by several laboratories. Eligible sources of clinical isolates included all sampled body sites as well as medical devices (e.g. catheters, prostheses). Isolates derived from the urinary tract were limited to 25% of the total number. Isolates (one per patient) were collected consecutively from patients with community-acquired or healthcare-associated infections. No restrictions were applied regarding patient age, gender, medical history or previous use of antimicrobials. Gram-negative isolates were also tested for susceptibility to amikacin (AMI), cefepime (CFP) and ceftazidime (CFM), whereas Gram-positive isolates were additionally tested for susceptibility to linezolid (LZD), penicillin (PEN), and vancomycin (VAN). S. pneumoniae isolates were additionally tested for susceptibility to azithromycin (AZI), clarithromycin (CLA), erythromycin (ERY) and clindamycin (CLI) at the central laboratory. In 2006, imipenem was tested instead of meropenem against the majority of collected isolates. Confirmation of ESBL production in E. coli and Klebsiella spp. isolates was performed according to CLSI guidelines using discs of cefotaxime (30 µg), cefotaxime-clavulanic acid (30/10 µg), ceftazidime (30 µg), and ceftazidimeclavulanic acid (30/10 µg) [26]. Antibiotic discs were manufactured by Oxoid, Inc. (Ogdensburg, NY, USA). Mueller-Hinton agar was produced by Remel, Inc. (Lenexa, KS, USA). All clinical isolates were sent to the central laboratory which organised the transport of the isolates and managed the study database. It also re-identified the isolates and verified the susceptibility results of 10-15% of the isolates annually. Quality control (QC) strains included S. aureus ATCC 29213, S. pneumoniae ATCC 49619, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853, H. influenzae ATCC 49247, and H. influenzae ATCC 49766. MIC data of the clinical isolates were only considered for evaluation if the MICs of the QC strains determined on the day of susceptibility testing were within the quality control ranges defined by CLSI [26]. QC strains used for quality control of ceftazidime and cefotaxime discs were K. pneumoniae ATCC 700603 (ESBL-positive) and E. coli ATCC 25922 (ESBL-negative), as well as P. aeruginosa (ATCC 27853). All isolate data were subject to a quality assurance programme to ensure the validity of the results. European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (version 5.0) were applied to antimicrobial agents and organisms for interpretation [27]. Neither EUCAST nor CLSI have set breakpoints for tigecycline against A. baumannii. Therefore, the breakpoints proposed by Jones et al. [28] (susceptible: ≤2 µg/mL; resistant: ≥8 µg/mL) were tentatively used for the determination of resistance rates when testing this organism/antibiotic combination.

Results
Between 2006 and 2014, the 25 participating laboratories collected a total of 36,044 isolates comprising 12,542 Gram-positive isolates and 23,502 Gram-negative organisms. Annual MIC 50 and MIC 90 values as well as resistance rates of tigecycline and comparators for the most clinically relevant species are shown in Table 1. A total of 30.1% of these isolates were collected from intensive care patients.
The percentage of meropenem-resistant strains among all Acinetobacter spp. isolates (n=893) increased from 4,2% in 2007 to 33% in 2014. Resistance to amikacin remained relatively stable at about 10% until 2012, but then increased to more than 30% in 2013/2014. A similar trend was observed for levofloxacin with resistance rates increasing from around 20% to ca. 40% in the last two years. MIC 50 and MIC 90 values of tigecycline were in the range of 0.12-0.25 µg/ml and 0.5-2 µg/ml, respectively. Applying the tentative breakpoints proposed by Jones et al. [28], none of the isolates tested were classified as tigecycline-resistant.

Escherichia coli
Among the 2,385 isolates, 16.7% (n=399) showed an ESBL-phenotype. The ESBL rate slightly increased during the test period, with annual rates ranging between 9.3% and 22.7% (Figure 1). Resistance to ceftriaxone and levofloxacin was lowest in the first year of the study period and then varied between 15-25% and 27-42%, respectively, but a clear trend was not observed for either drug.
Throughout the study, levofloxacin resistance was more common among ESBL-positive isolates (58-87%) than among ESBL-negative isolates (18-36%), as expected. Piperacillin-tazobactam resistance remained stable, with rates of <10% (data not shown) for ESBL-negative isolates and rates varying between 9% and 38% for ESBL-positive isolates. Meropenem susceptibility was high with no resistant isolates until 2012 and <1% in the last two years. Tigecycline was constantly active against E. coli during the entire study period, with MIC 90 values of 0.25-1 µg/ml and an overall susceptibility rate of 100%.

Enterobacter spp.
Meropenem activity against E. aerogenes (n=395) and E. cloacae (n=1,762) remained very high throughout the reporting period, with resistance rates ranging between 0% and 1.6% for both species. Low resistance rates were also observed for tigecycline (0-7% and 2-7%, respectively). Piperacillin-tazobactam was less active against either species, with resistance rates fluctuating around 20%.

Klebsiella spp.
The ESBL rates found for K. pneumoniae (n=1,481) ranged between 5.9% to 22.1%, with a mean of 15% (Figure 1). Levofloxacin and piperacillin-tazobactam showed poor activity against ESBL-positive isolates, with resistance rates of 50% and 29% at the end of the study period. In contrast, meropenem remained highly active against K. pneumoniae, though resistance was observed in 2008 and between 2011 and 2013, albeit at fairly low rates. The MIC 90 of tigecycline remained at ≤2 µg/ml throughout the reporting period (≤6% resistant isolates overall). However, six out of 38 ESBL-producing isolates exhibited resistance to tigecycline in 2014. As to K. oxytoca, there were 20 ESBL-positive strains among 829 isolates (2.4%). Resistance to meropenem was not detected throughout the reporting period. Tigecycline resistance rates were 0-4% even for ESBL-positive isolates (data not shown). Resistance rates for piperacillin-tazobactam and levofloxacin greatly varied during the study period, with approximately 20% and <10% resistant isolates, respectively, in the last year.

Stenotrophomonas maltophilia
S. maltophilia isolates (n=303) were collected in the last 3 years of the reporting period. MIC 50 and MIC 90 values recorded for tigecycline were 0.25 and 0.5-1 µg/ml, respectively. Minocycline was slightly less active than tigecycline, but the MIC 90 values were also 0.5-1 µg/ml (data not shown). MIC 90 values of levofloxacin were 2-4 µg/ml. Trimethoprim-sulfamethoxazole was not tested.
The proportion of vancomycin-resistant (VRE) strains among the E. faecium isolates increased from 7.4% in 2006 to 31.6% in 2014 ( Figure 1), but remained very low among E. faecalis isolates (data not shown). Resistance rates for levofloxacin were very high (90.7% overall) for E. faecium and about 40% for E. faecalis. Further, resistance for ampicillin was low in E. faecalis, while 92.4% of E. faecium isolates were found to be resistant. In contrast, 99-100% of the E. faecium isolates, including VRE, remained susceptible to tigecycline and linezolid throughout the reporting period.

Staphylococcus aureus
The proportion of MRSA among S. aureus isolates (n=2,351) varied over the years, with an average rate of 20.4% (range 14.3% to 50.7%) with higher and lower MRSA rates in the first an second half of the reporting period, respectively (Figure 1). Resistance to levofloxacin varied between 4.9% and 17.8% among MSSA isolates and between 73.3% and 98.1% among MRSA isolates.
All S. aureus isolates tested were susceptible to tigecycline, linezolid and vancomycin.

Streptococcus pneumoniae
The susceptibility pattern of S. pneumoniae remained largely unchanged during the observation period. Resistance rates for penicillin and ceftriaxone varied between 0% and 3.2% and those for levofloxacin between 0% and 2.7%, while the resistance rate for clarithromycin ranged from 8.5% to 22.5%, without a clear temporal trend. Resistance to linezolid was lacking and tigecyline showed MIC 50 and MIC 90 values of 0.015-0.03 µg/mL and 0.015-0.06 µg/mL, respectively.

Discussion
Given the rapidly changing landscape of antimicrobial resistance, particularly in Gram-negative bacteria [29], long-term monitoring of the activity of available antibiotics against common and problematic pathogens involved in serious infections is of great importance in the management of infectious diseases. MRSA rates have been reported to decline in Germany in recent years [11], [30], [31], [32], while the rates of Gram-negative bacteria producing ESBLs and the rate of VRE remained either unchanged or increased over the past years [11], [31], [32], [33]. Efficient hygiene measures, antibiotic stewardship programmes and rational use of the available treatment options are crucial for the maintenance of the ability to control serious bacterial diseases, particularly in critically ill patients. Given the highly heterogenerous healthcare situations in Europan countries, regional longitudinal susceptibility data are key to enable adequate 14 early action and policy adjustments if untoward trends in resistance emerge. The present study provides data from a larger and more diverse sample of isolates than previous surveys on the comparative susceptibility of tigecycline in Germany [12], [13], [14], [24]. The moderate increase in the prevalence of ESBL-producing E. coli and the stable proportion of ESBL-producing K. pneumoniae isolates found in the present study ( Figure 1) corresponds approximately with the ESBL rates reported by the Paul-Ehrlich-Gesellschaft für Chemotherapie (PEG) [31]. The low ESBL rate observed in K. oxytoca is explained by the low activity of the K1 beta-lactamase against the antibiotics cefotaxime and ceftazidime [34], which were used for ESBL screening. The rate of carbapenem-resistant Acinetobacter spp. showed a dramatic increase, reaching 33% in 2014, which is consistent with observations of other German surveillance studies for A. baumannii [31], [35]. Regarding Gram-positive pathogens, Gastmeier et al. [29] reported a strong increase in the proportion of VRE among nosocomial infections in Germany for the time period of 2007 to 2012. This observation corresponds to the trend towards higher VRE rates in the second half of the present study. MRSA rates varied considerably during the study period. The unusually high percentage of MRSA observed in 2009 is most likely due to random variation, given the low total number of isolates tested in that year. The downward trend of MRSA rates in the last three years, as also observed by other study groups in Germany, possibly reflects improved effects of infection control measures. The clinical usefulness of tigecycline in the management of complicated infections, particularly cIAI and cSSTI, including those caused by pathogens with MDR has recently been confirmed in a large observational study programme performed in Germany [36], [37], [38]. These observations are supported by the results of the present multicentre in vitro study comprising more than 36,000 clinical isolates obtained from patients with community-acquired or nosocomial infections in Germany, confirming that eight years after its introduction into the German market, tigecycline invariably retains its high antimicrobial activity against a broad range of important Gram-negative and Gram-positive pathogens, including ESBL-producing Enterobacteriaceae, MRSA and VRE. The good activity of tigecycline found in the present study reassured the results of a German Tigecycline Evaluation and Surveillance Trial (G-TEST) performed between 2005 and 2009 [10], [11], [12]. Moreover, the German long-term findings for tigecycline from TEST are consistent with those published for Europe in general [39], and other individual European regions, particularly France [15], Italy [16], and Eastern European countries [17]. MIC 50 and MIC 90 values of 0.25 µg/mL and 0.5-1 µg/mL, respectively, assessed for S. maltophilia in the present study point to a potential usefulness of tigecycline in the mangement of infections caused by this opportunistic MDR pathogen of growing importance [40].
Tigecycline has been shown to be effective and well tolerated at higher than standard doses in critically ill patients infected with MDR bacteria [41] and in patients with hospital-acquired pneumonia [42]. Further studies investigating higher dosages of tigecycline in severly ill patients with difficult-to-treat infections appear warranted. In conclusion, our findings indicate sustained activity of tigecycline against pathogens known to cause infections in severely ill patients. This is true for isolates susceptible to standard antibiotics as well as MDR bacteria like ESBLproducing Enterobacteriaceae, carbapenem-resistant Acinetobacter spp., VRE and MRSA, where choices of active drugs are generally limited or resistance rates worrisome.
Notes K.B. has received grants or research support from from the Bundesministerium für Bildung und Forschung (BMBF), Germany, the German Center for Infection Research (DZIF), the Bundesministerium für Wirtschaft (BMWi), the European Territorial Cooperation (ETC)/IN-TERREG, Cepheid and Pfizer as well as lecture, travel and other fees from Cepheid, Cubist Pharmaceuticals, MSD Sharp & Dohme, Novartis Pharma, Oxoid, Pfizer and Siemens Healthcare Diagnostics.